Improved hLigI inhibition activity of Curcumin Monocarbonyl Analogue-Dithiocarbamate Hybrids: Design, Synthesis and Biology

doi:10.21203/rs.3.rs-1654597/v1

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Improved hLigI inhibition activity of Curcumin Monocarbonyl Analogue-Dithiocarbamate Hybrids: Design, Synthesis and Biology

https://doi.org/10.21203/rs.3.rs-1654597/v1

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Human DNA ligase (hLigI) plays an important role in the process of DNA replication and repair, making it an important target for cancer. In our tireless efforts for finding selective inhibitors against hLigI, this article reports the design, synthesis and biological activity of a series of 71 curcumin-monocarbonyl-dithiocarbamate hybrid molecules. Many of the synthetic compounds tested in this study show a significant inhibitory effect on the human DNA ligase 1 (hLig1) activity (> 90% inhibition) and demonstrated significant anti-proliferative activity against colon cancer cells (DLD-1) in a concentration dependent manner. In addition, at a concentration of 10 µM, certain compounds showed selective activity towards DLD-1 cells as compared to the normal HEK-293 cells. Overall, our studies demonstrate that two compounds in this series (27 and 49) have significantly better DNA ligase 1 inhibition and anti-proliferative activities than the previously reported monocarbonyl-curcumin hybrid (compound 23*). This study brings us closer towards our quest to explore the molecular space for novel hLig1 inhibitors and to find inhibitors that will be suitable for clinical trials in cancer patients.

hLigI inhibitor

antiproliferative activity

DNA ligase activity

dithiocarbamate

Accurate, error free DNA replication and repair process are essential for cell survival and its progression [1–3]. The DNA replication is a multistep process that requires the involvement of multiple proteins [4, 5]. DNA ligases are the essential proteins that seal the nick between DNA strands at the final step of DNA replication and repair [6, 7]. In humans and other eukaryotes, three different types of ligases are reported, i.e. human DNA ligase I (hLigI), human DNA ligase III (hLigIII) and human DNA ligase IV (hLigIV), which require ATP as a cofactor for the completion of DNA ligation [8–13]. Among them, hLigI has been widely studied and its crystal structure with DNA is available [PDB ID-1X9N] [14]. Notably, hLigI protein is not only the major DNA replicative ligase but also plays important roles in the repair pathway of single strand DNA breaks [15, 16]. Inhibition or deficiency of hLigI causes several abnormalities such as growth retardation, solar sensitivity and immunodeficiency, which indicates the importance of hLig1p for a healthy life [17–20]. The hLigIp expression is reported elevated in several cancers and in rapidly dividing cells [21, 22]. These studies suggest that hLigI can serve as a companion target for the development of novel and specific cancer therapy. We have used several approaches such as in silico based virtual screening and molecular hybridization for the identification of antiligase compounds. Although many compounds belonging to different chemical families have shown excellent antiligase and anticancer activity, however none has reached the clinical trials [23–32, 11, 33]. Therefore, the need for new compounds that can exhibit not only potent antiligase activity but also possess drug-like properties for therapeutic efficacy still exists.

In our previous research [29], we generated a pharmacophore model from the previously reported DNA ligase inhibitors and performed virtual screening of a natural product library. Our screening indicated that Curcumin could potentially inhibit the DNA ligase 1 activity. We then went on to synthesize a series of monocarbonyl-curcumin-thiourea hybrids and checked their activity. We reported the best compounds that enhanced the hLig1 inhibition and antiproliferative activity as compared to curcumin itself [29].

In this study, we further extend this research to report more potent curcumin derivatives by synthesizing a novel series of fifty-nine curcumin-mono-carbonyl-Dithiocarbamates (Cur-DTC) and evaluated their potential to inhibit hLig1 nick sealing activity followed by their cytotoxicity and antiproliferative activity in colorectal cancer cells (DLD-1). Since there are not many new chemical entities for this series of compounds in the literature, we used an in silico approach to discover a new lead compound, which was the first inhibitor related to curcumin monocarbonyl family that was found to inhibit the DNA nick sealing activity of hLigIp. Our aim was also to broaden the chemical space in order to design the best possible hLig1 inhibitor with highly specific and desirable biological activity.

2.1 Chemistry

Scheme 1 illustrates the general procedure for the synthesis of (3E,5E)-substituted 3,5-bis (substituted benzylidene)-4-oxopiperidine-1-carbodithioate (5–52). In the first step, (3E,5E)–3,5–bis (substituted benzylidene) piperidin–4–one (3a-3k) were synthesized through double aldol condensation of different substituted benzaldehydes (1a-1k) and 4–piperidinone hydrochloride hydrate (2) in acidic condition. The reaction was carried out with good yield at room temperature. The precipitated yellow solid was filtered and then reacted with potassium carbonate in acetone:water (5:1) for 2 hours to yield 3a–3k (Scheme 1) [29]. These 2^o amines 3a-3k when treated with carbon disulfide and aq. NaOH in DCM at 0^oC yielded novel intermediate sodium salts 4a-4k. These salts were reported and evaluating against hLig protein for the first time. Furthermore, 4a–4k were reacted with different substituted alkyl/benzyl/phenacycl bromides in water:acetone (5:1) at 10–15 ^oC for 1–2 hour yields compounds 5–52 in good yields.

Reagents and conditions: (a) Glacial acetic acid, conc. HCl, RT, 48 h; (b) K₂CO₃, acetone:water (5:1), 20 ^oC, 2 h; (c) carbon disulfide (1.3 equiv), aqNaOH, DCM, 0 ^oC, 2 h; (d) R-Br (1.2 equiv.), water:acetone (5:1), 10-15^oC, 1–2 h.

2.2 Biological Evaluation

Antiligase and antiproliferative activity of synthesized compounds (5–52)

We synthesized eleven Sodium (3E,5E)-3,5-bis(substituted benzylidene)-4-oxopiperidine-1-carbodithioates (4a-4k) and forty-eight (3E,5E)-substituted-3,5-bis (substituted benzylidene)-4-oxopiperidine-1-carbodithioates (hereinafter referred to as curcumin-DTC hybrids) and evaluated them for inhibition of hLig1 nick sealing activity and antiproliferative activity in colon cancer (DLD-1 cells) (Table 1). We varied the substitutions at R₁ and R₂ to study the activity patterns of different electron donating and withdrawing substitutions and varied the alkyl group on position R₂ as well. Activity was tested in colon cancer since our earlier study showed that the compounds were most active against colon cancer as compared to other cancer cells.

At R1-position, regardless of the electron-donating or electron-withdrawing group, substitution at 4th -position of benzene ring (R1) yields more active compounds, except 4-methoxy substitution (4-Cl, 4-F, 4-Me, 4-iPr). Any modification at this position resulted in either decreased (2-OMe, 3-Me, 2-F, 2-Me) activity or resulted in complete loss of activity (3,4-dimethoxy).

Among all the favorable substitutions at 4th position, fluoro group yielded good antiligase activity and showed antiproliferative activity against DLD1 cells at 10 µM concentration. We also found that the substitutions at 2nd and 3rd positions yielded the desired activity in which the 2-methoxy group (3 compounds) has shown > 90% hLigI inhibition (at 50 µM concentration) and > 50% antiproliferative activities at 10 µM concentration. However, none of the compounds with substations 4-iPr, 4-OMe, 3,4-di-OMe, 2-Me could show > 50% antiproliferative inhibition in DLD1 cells. Here the substitution at R1 did not show any obvious effect, but the substitution of electron-withdrawing groups at R2 resulted in three active compounds rather than substitution with electron-donating groups. Substitution of R2 has also been found to be crucial for activity, because the aromatic ring-containing groups showed increased activity (i.e., benzyl, phenacyl and methoxy-phenacyl groups) compared to alkyl substitutions (i.e., allyl and methyl groups).

From this initial screen we shortlisted thirteen compounds that showed at least 90% inhibition of hLig1 mediated DNA ligase activity and also demonstrated cellular cytotoxicity of 50% in DLD-1 cells (Table 1; compounds 4d, 4e, 4k, 7, 16, 17, 26, 27, 29, 32, 35, 37, and 49). These were further screened at lower concentrations and comparative cytotoxicity was ascertained against the normal human HEK-293 cells.

Table 1

Synthesized compounds were tested for their hLig1 enzymatic inhibition and cytotoxicity against DLD1 cells. Only those compounds that showed > 90% hLig1 inhibition at 50µM concentration were tested for their cytotoxicity against DLD1 cells.
Comp. No.	% inhibition of hLig1 activity at 50 µM concentration	% antiproliferative activity in DLD1 cells at 10 µM
4a	22.36	ND
4b	22	ND
4c	19.03	ND
4d	92.32	52.17
4e	91.84	49.72
4f	0	ND
4g	99.05	32.63
4h	16.12	ND
4i	99.16	20.31
4j	91	38.26
4k	99.9	51.67
5	81.84	ND
6	71.99	ND
7	97.16	58.32
8	76.92	ND
9	97.15	7.32
10	85.21	ND
11	97.26	7.57
12	16.78	ND
13	14.07	ND
14	17.7	ND
15	0	ND
16	99.32	50.9
17	97.26	54.4
18	47.92	ND
19	73.83	ND
20	69.04	ND
21	0	ND
22	7.28	ND
23	3.44	ND
24	0	ND
25	42.61	ND
26	89.43	52.81
27	99.13	50.6
28	99.2	43.56
29	99.49	56.4
30	96.16	8.84
31	96.44	13.48
32	99.85	59.74
33	98.65	2.57
34	98.96	17.32
35	91.2	56.32
36	98.63	3.86
37	96.34	51.88
38	89.1	1.23
39	93.85	9.63
40	99.49	7.32
41	50.5	ND
42	41.59	ND
43	18.33	ND
44	17.02	ND
45	0	ND
46	65.99	ND
47	54.05	ND
48	46.42	ND
49	93.2	64.2
50	0	ND
51	0	ND
52	63.35	ND

The shortlisted compounds were tested for hLig1 inhibition and antiproliferative activity at 10 µM concentrations. Table 2 lists those compounds (4e, 17, 27, 29, 37, and 49) that showed better antiligase as well as antiproliferative activity than our previous lead molecule (Mandalapu, Singh, et al., 2016). Among these selected compounds, we observed that 4-F, 3-Me substitution at R1 and benzyl, methoxy-phenacyl groups at R2 showed good activity profile, but methoxyphenacyl substitution was found to be toxic against HEK-293 cells. However, the benzyl substitution at R2 (27 and 49) showed selective activity against colon cancer cells (DLD-1). Therefore, these compounds (27 and 49) were further tested for dose-dependent antiligase and antiproliferative activities.

Table 2

*In vitro* antiligase activity and antiproliferative activity of shortlisted compounds against DLD-1 (Colon cancer cell line) and normal HEK-293 (Human embryonic kidney) cell line.
Compound No.	% Inhibition of Ligase activity at 10µM	Cytotoxicity in DLD1 cells at 10µM	Cytotoxicity in HEK-293 cells at 10µM
Compound 35	37.86 ± 4.11	51.83 ± 4.50	49.9 ± 6.6
Compound 37	56.75 ± 8.07	59.2 ± 7.39	33.05 ± 4.35
Compound 7	41.97 ± 9.51	53.26 ± 5.05	66.67 ± 14.2
Compound 32	39.07 ± 8.09	54.25 ± 5.49	39.9 ± 0.62
Compound 27	68.49 ± 9.67	64.92 ± 4.68	29.85 ± 0.55
Compound 29	57.74 ± 4.86	66.58 ± 10.18	35.8 ± 1.6
Compound 26	37.63 ± 1	68.4 ± 15.5	40.97 ± 1.47
Compound 4e	45.37 ± 12.83	54.02 ± 4.3	53.17 ± 14.32
Compound 4d	36.47 ± 6.66	67.23 ± 15.06	53.15 ± 12.51
Compound 16	39.29 ± 4.58	67.15 ± 16.25	44.14 ± 7.48
Compound 17	49.14 ± 13.85	63.77 ± 9.3	42.05 ± 4.45
Compound 4k	38.80 ± 5.56	57.86 ± 6.19	61.99 ± 8.51
Compound 49	91.31 ± 4.87	67.4 ± 3.2	30.77 ± 2.3
Compound 23* [29]	31.11 ± 2.20	50.88 ± 5.09	37.93 ± 1.75

The hLigI inhibition was tested in a ligation assay with purified protein and nicked ligatable DNA substrate labeled with FAM1. As shown in Fig. 1, increasing the concentration (2.5, 5, 10 and 20 µM) of compounds (27, 49 and 23*) decreased the hLig1 mediated ligated product in a dose dependent manner. The amount of unligated product can be seen to increase as we increase the dose of inhibitors. We quantified the ligated products by densitometry and represented the data in a graph. The results show that compounds 27 and 49 with IC₅₀ values (for antiligase activity) of 2.19 and 3.9 µM were almost three times better than the ligation inhibitory activity of previously reported compound i.e compound 23* (IC₅₀ value 18.57 µM), however compound 49 show best antiligase activity among all.

Figure 1

The left panel shows the gel images for dose dependent antiligase activity of compounds 49, 27 and the previously reported compound 23* against hLigIp. Right panel shows the quantified antiligase activity of compounds in a graph.

Cellular activity of compounds 27 and 49 against DLD-1 and HEK-293 cells

We performed dose-dependent (0.6 1.2, 2.5, 5, 10, 20 µM) cellular activity of compounds 27, 49 and compound 23* in DLD-1 and HEK-293 cells. The graphs represented in Fig. 2A-C, clearly show that all compounds exhibit dose-dependent antiproliferative activity. Compared with our previous lead molecule (compound 23* with IC₅₀ = 11.6 µM), both compounds 27 and 49 from this novel library have better antiproliferative activity with IC₅₀ of 3 and 3.8 µM respectively in DLD1 cells. We also evaluated the cytotoxicity of compounds in HEK-293 cells and found that compound 27, 49 and 23* have IC₅₀ values of 6.6, 27.4 and 29.09 µM respectively. The data suggests that both compounds 27 and 49 show better cellular activity than compound 23*. However, compound 27 was found to be more toxic against HEK-293 cells as compared to compound 23*. Therefore, compound 49 is our choice of lead molecule.

We studied the morphological change in DLD-1 cells induced by new compounds 27 and 49. The cells were treated with compounds at their IC₅₀ values for 24 and 48h and the bright field image was captured by microscope. Figure 2D shows that both compounds promote cell atrophy, cell detachment from the surface and growth arrest, which propose that both compounds promote cellular death. We studied the effect of compounds on antiproliferative activity of cells with the help of clonogenic cell survival assay. The Fig. 2E, shows that the increasing concentration of compounds (0.75, 1.5 and 3 µM) gradually decreased the number of colonies. The data suggests that both compounds were able to abrogate cell proliferation and resulted in a reduction in the number of DLD1 cell colonies.

2.3 In silico Binding Study of Compounds with hLigI Protein

In order to model the binding of identified inhibitors with hLigI, we have analyzed the docking complexes of these inhibitors with the crystal structure of human DNA ligase I (PDB ID-1X9N). The docking scores obtained are quite reasonable and the most active compound 49 has a better docking score against the DNA binding domain (DBD) of hLigI, which may be able to explain its potent antiligase activity. The compounds dock nicely into the DBD domain of hLig1, suggesting that it is related to DNA binding. Figure 3 shows the binding pattern of the docked complex of the most active compound with highest antiligase activity. As can be seen from Fig. 3, the oxygen attached to the piperidine ring is close to the DNA binding residue Leu 338 and forms a hydrogen bond. Another hydrogen bond is formed with Gly 339 with the same atom. The interactions of these residues with the compound are concordant with the previous studies revealing that these protein residues are known to play an important role in the binding of hLigIp to DNA (Bathula et al., 2018; Chen et al., 2008; Zhong et al., 2008). We may conclude that the interactions of compound 49 with hLigIp may be responsible to increased stability of the docked complex and may explain the effective hLigI inhibition observed for compound 49 in in vitro assays. The glide docking scores of all the compounds are provided in Table 2. Glide Score is an empirical scoring function that approaches the ligand binding free energy. It is an ensemble of many terms, including contributions from electrostatic and van der Waals force fields and terms rewarding or penalizing interactions known to influence ligand binding. Since it simulates the binding free-energy, more negative values translate into stronger binding.

Table 2

Glide docking scores for tested hLig1 inhibitors.
S.No.	Compounds	Docking Score (Glide)
1.	Compound 35	-2.56
2.	Compound 37	-3.21
3.	Compound 7	-3.79
4.	Compound 32	-2.87
5.	Compound 27	-4.05
6.	Compound 29	-4.11
7.	Compound 26	-2.36
8.	Compound 4e	-3.82
9.	Compound 4d	-2.73
10.	Compound 16	-2.06
11.	Compound 17	-4.03
12.	Compound 4k	-2.81
13.	Compound 49	-4.43

2.4 Cell cycle analysis of DLD-1 after treatment with compound 49

In the terms of protein and cell based activity and safety index, compound 49 was found more promising than compound 27, therefore we conducted further studies with compound 49. We studied the effect of compound 49 on the cell cycle progression of DLD-1 cells. The DLD-1 cells were synchronized at G0/G1 phase by serum starvation and treated with compound 49 at 3 µM (half IC₅₀) concentration for different time periods. The results (Fig. 4) show the increased accumulation of cells at the G2/M phase over the treatment time. Since the ligases play their role at the last part of DNA replication and repair pathway, therefore it was expected for ligase inhibitors to arrest the cell cycle at G2/M phase.

In this study, we report the synthesis and biological evaluation (antiligase and selective cytotoxicity) of curcumin-DTC hybrid compound library of 71 molecules. Eleven Sodium (3E, 5E)-3,5-bis(substituted benzylidene)-4-oxopiperidine-1-carbodithioates (4a-4k) and forty-eight (3E,5E)-substituted-3,5-bis(substituted benzylidene)-4-oxopiperidine-1-carbodithioates are reported. We first evaluated the potency of these compounds for DNA ligase 1 inhibition at 50 µM concentration and cellular cytotoxicity at 10 µM concentration against DLD-1 (colon cancer) cells. We identified a new derivative called compound 49 that demonstrated 3X better antiligase activity and selective cytotoxicity than our earlier lead molecule (compound 27*) reported in Mandalapu and Singh, et al., 2016.

The newly identified compound 49 inhibited the colony-forming ability and antiproliferative activity of DLD-1 cells in a dose dependent manner. DNA ligation assay, cell viability assay and docking studies have shown that among the two new derivatives, compound 49 has the highest activity. Compound 49 also showed G2/M phase arrest of the cell cycle of DLD-1 cells. Finally, this study strongly supports the maturity of our synthetic approach, which is evident from the synthesis of highly active and specific compounds based on our previous study. Considering these results, it may soon be possible to identify specific antiligase molecules that will be suitable for clinical studies.

Chemistry

In general, all reagents and solvents were obtained from commercial vendors and were used without further purification. All reactions were monitored by thin-layer chromatography (TLC) using F₂₅₄ silica gel plates with fluorescence (Aldrich). Melting points were determined in open capillary tubes on an electrically heated block and are uncorrected. IR spectra (ν_max in cm^− 1) of the compounds were recorded on Perkin Elmer FT-IR RX1 PC spectrophotometer. ¹H NMR spectra were recorded on Bruker Supercon Magnet Avance DPX-200/DRX-300 spectrometers (operating at 500, 400 and 100 MHz, respectively, for ¹H and ¹³C) in deuterated solvents with TMS as internal reference (chemical shifts δ in ppm, J in Hz.). Electrospray ionization mass spectra (ESI-MS) were recorded on Ion Trap LCQ Advantage Max-IT (Thermo Electron Corporation). High-resolution mass spectra (HRMS) were recorded on a 6520 Agilent Q TofLC MS/MS (accurate mass). Elemental analyses were performed on a Carlo Erba EA-1108 micro analyzer/Vario EL-III C, H, N analyzer. All compounds were analyzed of C, H, N and the results obtained were within ± 0.4% of calculated values. Intermediate sodium (3E,5E)-3,5-bis(substituted benzylidene)-4-oxopiperidine-1-carbodithioates 4a-4k were synthesized according to our previous reported procedure.

Sodium (3E,5E)-3,5-dibenzylidene-4-oxopiperidine-1-carbodithioate(4a):

The title compound was synthesized from (3E,5E)-3,5-dibenzylidenepiperidin-4-one (1 eq., 3a), AqNaOH (2 eq.,) and carbon disulfide (2 eq.,) in 73% yield as reddish solid.

IR (KBr) ν (cm^− 1): 3019, 2401, 1637, 1215; ¹H NMR (400 MHz, DMSO-d6): δ7.62–7.60 (6H, m), 7.51–7.47 (4H, m), 7.44–7.40 (2H, m), 5.84 (4H, s); ¹³C NMR (400 MHz, DMSO): δ215.4, 187.7, 136.4, 135.5, 134.3, 134.4, 134.2, 130.9, 130.9, 129.6, 129.5, 129.2, 129.1, 50.7;

Sodium (3E,5E)-3,5-bis(4-methoxybenzylidene)-4-oxopiperidine-1-carbodithioate (4b):

IR (KBr) ν (cm^− 1): 3021, 2400, 1612, 1413, 1215, 1114; ¹H NMR (400 MHz, DMSO): δ 7.58–7.55 (6H, m), 7.05 (4H, d, J = 8.84 Hz), 5.82 (4H, s), 3.82 (6H, s); ¹³C NMR (400 MHz, DMSO): δ 215.1, 187.4, 160.5, 135.2, 132.9, 132.2, 128.0, 114.7, 55.7, 50.8;

Sodium (3E,5E)-3,5-bis(3,4-dimethoxybenzylidene)-4-oxopiperidine-1-carbodithioate (4c):

IR (KBr) ν (cm^− 1): 3019, 2401, 1654, 1215, 1119; ¹H NMR (400 MHz, DMSO): δ7.56 (2H, s), 7.27–7.26 (2H, m), 7.19 (2H, d/d, J₁ = 11.8 Hz, J₂ = 8.52 Hz), 7.07 (2H, d, J = 8.48 Hz), 5.84 (4H, s), 3.82–3.82 (12H, m), ¹³C NMR (400 MHz, DMSO): δ215.1, 187.2 150.3, 148.9, 135.7, 132.2, 128.1, 124.7, 114.5, 112.1, 56.0, 56.0, 50.8;

Sodium (3E,5E)-3,5-bis(2-methoxybenzylidene)-4-oxopiperidine-1-carbodithioate (4d):

IR (KBr) ν (cm^− 1): 3019, 1647, 1403, 1215, 1070; ¹H NMR (400 MHz, DMSO-d₆): δ7.83 (2H, s), 7.51 (2H, d, J = 6.72 Hz), 7.43–7.39 (2H, m), 7.10 (2H, d, J = 8.12 Hz), 7.03 (2H, t, J = 7.48 Hz), 5.71 (4H, s), 3.86 (6H, s); ¹³C NMR (100 MHz, DMSO-d₆):δ215.0, 187.6, 158.5, 133.7, 131.4, 130.9, 130.5, 123.9, 120.7, 111.7, 56.0 50.8;

Sodium (3E,5E)-3,5-bis(2-fluorobenzylidene)-4-oxopiperidine-1-carbodithioate (4e)

IR (KBr) ν (cm^− 1): 3019, 1646, 1403, 1215; ¹H NMR (400 MHz, MeOD): δ7.88 (2H, s), 7.69–7.65 (2H, m), 7.48–7.42 (2H, m), 7.30–7.26 (2H, m), 7.22–7.17 (2H, m), 5.78 (4H, s); ¹³C NMR (100 MHz, DMSO-d₆):δ215.5, 187.0, 162.2, 159.8, 135.7, 132.0, 131.9, 131.3, 131.3, 127.8, 127.7, 125.1,125.0, 123.0, 122.9, 116.2, 116.0, 50.7;

Sodium (3E,5E)-3,5-bis(4-fluorobenzylidene)-4-oxopiperidine-1-carbodithioate (4f):

3019, 2401, 1637, 1404, 1215; ¹H NMR (400 MHz, DMSO-d6): δ 7.68–7.66 (4H, m), 7.65 (2H, s), 7.32 (4H, t, J = 8.72 Hz), 5.80 (4H, s); ¹³C NMR (100 MHz, DMSO- d6): δ215.4, 187.5, 164.0, 161.5, 136.1, 134.4, 134.1, 134.0, 133.3, 133.2, 133.2, 133.1, 133.1, 131.9, 131.9, 116.3, 116.0, 50.6;

Sodium (3E,5E)-3,5-bis(4-chlorobenzylidene)-4-oxopiperidine-1-carbodithioate (4g):

IR (KBr) ν (cm^− 1): 3021, 1612, 1215, 670; ¹H NMR (400 MHz, DMSO-d6): δ 7.72 (2H, s), 7.59 (4H, d, J = 8.50 Hz), 7.46 (4H, d, J = 8.50 Hz), 5.83 (4H, s); ¹³C NMR (100 MHz, DMSO-d6): δ215.3, 187.3, 134.8, 134.4, 134.3, 134.1, 132.6, 129.1, 50.7;

Sodium (3E,5E)-3,5-bis(4-methylbenzylidene)-4-oxopiperidine-1-carbodithioate (4h):

Yellow solid (80%); mp 180-182^oC; IR (KBr) ν (cm^− 1): 3021, 2402, 1607, 1215, 761, 669; ¹H NMR (400 MHz, DMSO): δ7.56 (2H, s), 7.49 (4H, d, J = 8.12 Hz), 7.30 (4H, d, J = 7.96 Hz), 5.82 (4H, s), 2.36 (6H, s); ¹³C NMR (400 MHz, CDCl₃): δ215.1, 187.6, 139.6, 135.6, 133.5, 132.5, 131.0, 129.8, 50.8, 21.4;

Sodium (3E,5E)-3,5-bis(4-isopropylbenzylidene)-4-oxopiperidine-1-carbodithioate (4i):

IR (KBr) ν (cm^− 1): 3025, 2401, 1612, 1215; ¹H NMR (400 MHz, MeOD): δ7.62 (2H, s), 7.42 (4H, d, J = 6.56 Hz), 7.23 (4H, d, J = 6.60 Hz), 5.75 (2H, d, J = 1.44 Hz), 2.88–2.80 (2H, m), 1.17 (12H, d, J = 5.52 Hz); ¹³C NMR (100 MHz, MeOD): δ213.8, 188.2, 150.5, 137.0, 132.5, 131.8, 130.7, 126.4, 51.4, 33.9, 22.8;

Sodium (3E,5E)-3,5-bis(2-methylbenzylidene)-4-oxopiperidine-1-carbodithioate (4j):

IR (KBr) ν (cm^− 1): 3020, 1620, 1215; ¹H NMR (400 MHz, MeOD): δ7.95 (2H, s), 7.43 (2H, d, J = 6.80 Hz), 7.28–7.24 (6H, m), 5.64 (4H, s), 2.38 (6H, s); ¹³C NMR (125 MHz, MeOD):δ213.6, 188.3, 137.9, 136.4, 133.9, 132.8, 129.9, 128.9, 128.8, 125.4, 51.2 18.8;

Sodium (3E,5E)-3,5-bis(3-methylbenzylidene)-4-oxopiperidine-1-carbodithioate (4k):

IR (KBr) ν (cm^− 1): 3020, 1646, 1403, 1215; ¹H NMR (400 MHz, MeOD): δ7.73 (2H, s), 7.43 (2H, s), 7.40–7.32 (4H, m), 7.23 (2H, d, J = 6.8 Hz), 5.85 (4H, s), 2.40 (6H, s); ¹³C NMR (100 MHz, DMSO-d6): δ215.2, 187.6, 138.3, 135.8, 135.2, 134.1, 131.6, 130.3, 129.0, 127.9, 50.9, 21.4;

Procedure for the synthesis of (3E,5E)-substituted 3,5-bis(substituted benzylidene)-4-oxopiperidine-1-carbodithioates (5–52)

The intermediates, sodium (3E,5E)-3,5-bis(substituted benzylidene)-4-oxopiperidine-1-carbodithioates (4a-4k, 1.3 equiv.) were dissolved in water:Acetone (5:1) at 10-15^oC and stirred for 5 min. Into the above stirred reaction mixture, We added different alkyl/aralkyl/phenacyl bromides (1 equiv.) in acetone (1mL) and stirred it for 1–2 hours. Reaction progress was monitored by TLC (30–40% EtOAC/Hexane), stopped the reaction upon completion and added 30 mL ethylacetate into the reaction. Stirred it for 5 min and the reaction mixture was further washed with excess water (3x20 mL) and the organic layer was separated, dried over Na₂SO₄ and evaporated in vacuo to afford yellow solids. They were further recrystallized from absolute ethanol to afford pure final (3E,5E)-substituted 3,5-bis(substituted benzylidene)-4-oxopiperidine-1-carbodithioates (5–52) in good yields.

(3E,5E)-allyl 3,5-dibenzylidene-4-oxopiperidine-1-carbodithioate (5):

Yellow solid (85%); mp: 180-182^oC; IR (KBr) ν (cm^− 1): 3021, 2402, 1617, 1509, 1216; ¹H NMR (400 MHz, CDCl₃): δ 7.90 (2H, s), 7.49–7.46 (10H, m), 5.89–5.78 (1H, m), 5.41 (4H, bs), 5.27–5.21 (1H, m), 5.14–5.10 (1H, m), 3.93–3.90 (2H, m); ¹³C NMR (400 MHz, CDCL₃): δ197.2, 186.5, 139.1, 134.3, 132.1, 130.3, 129.7, 128.8, 118.7, 50.8, 40.6; ESI-MS: m/z 392 (M + H⁺).

(3E,5E)-2-oxo-2-phenylethyl 3,5-dibenzylidene-4-oxopiperidine-1-carbodithioate (6):

Yellow solid (70%); mp 178-180^oC; IR (KBr) ν (cm^− 1): 3021, 2401, 1676, 1616, 1215; ¹H NMR (400 MHz, CDCL₃): δ 8.02 (2H, d, J = 7.16 Hz), 7.91 (2H, s), 7.49–7.43 (13H, m), 5.44 (4H, bs), 4.79 (2H, s); ¹³C NMR (400 MHz, CDCL₃): δ196.1, 192.9, 186.3, 139.3, 136.1, 134.2, 133.5, 130.3, 129.8, 128.9, 128.4, 58.94, 45.0; ESI-MS: m/z469 (M + H⁺); HRMS (ESI): m/z calculated for C₂₈H₂₃NO₂S₂ + H⁺ (M + H⁺): 470.1248. Found: 470.1244.

(3E,5E)-2-(4-methoxyphenyl)-2-oxoethyl-3,5-dibenzylidene-4-oxopiperidine-1-carbodithioate (7):

Yellow solid (79%); mp 173-175^oC; IR (KBr) ν (cm^− 1): 3019, 2401, 1675, 1605, 1510, 1216, 1037; ¹H NMR (400 MHz, CDCL₃): δ 7.99 (2H, d, J = 4 Hz), 7.90 (2H, s), 7.47–7.42 (10H, m), 6.93 (2H, m, J = 9Hz), 5.43 (4H, bs), 4.75 (2H, s), 3.87 (3H, s); ¹³C NMR (100 MHz, CDCL₃): δ196.3, 191.4, 186.4, 163.8, 139.3, 134.2, 130.8, 130.3, 129.8, 128.8, 113.8, 55.4, 50.8, 44.8; ESI-MS: m/z500 (M + H⁺); HRMS (ESI): m/z calculated for C₂₉H₂₅NO₃S₂ + H⁺ (M + H⁺): 500.1358. Found: 500.1347.

(3E,5E)-allyl 3,5-bis(4-methoxybenzylidene)-4-oxopiperidine-1-carbodithioate (8):

IR (KBr) ν (cm^− 1): 3020, 2401, 1602, 1511, 1216, 1030; ¹H NMR (400 MHz, CDCl₃): δ 7.85 (2H, s), 7.46 (4H, d, J = 8.28 Hz), 7.01 (4H, d, J = 8.76 Hz), 5.92–5.81 (1H, m), 5.41 (4H, bs), 5.26 (1H, d/d, J₁ = 1.32 Hz, J₂ = 16.96 Hz), 5.13 (1H, d, J = 10 Hz), 3.95 (2H, d, J = 6.96 Hz), 3.89 (6H, s); ¹³C NMR (400 MHz, CDCl₃): δ197.0, 186.1, 160.8, 138.5, 132.5, 132.3, 128.2, 127.1, 118.7, 114.4, 55.4, 51.0, 40.7; ESI-MS: m/z452 (M + H⁺);

(3E,5E)-benzyl 3,5-bis(4-methoxybenzylidene)-4-oxopiperidine-1-carbodithioate (9):

Yellow solid (82%); mp 176-180^oC; IR (KBr) ν (cm^− 1): 3021, 2402, 1619, 1216; ¹H NMR (400 MHz, CDCl₃): δ7.84 (2H, s), 7.46–7.44 (4H, m), 7.32–7.29 (5H, m), 6.99 (4H, d, J = 8.76 Hz), 5.40 (4H, bs), 4.53 (2H, s), 3.87 (6H, s); ¹³C NMR (100 MHz, CDCl₃): δ197.2, 186.3, 160.8, 138.5, 135.8, 132.5, 129.3, 128.5, 128.2, 127.5, 127.1, 114.4, 55.3, 50.8, 42.5; ESI-MS: m/z502 (M + H⁺);

(3E,5E)-2-oxo-2-phenylethyl 3,5-bis(4-methoxybenzylidene)-4-oxopiperidine-1-carbodithioate (10):

Yellow solid (80%); mp 168-170^oC; IR (KBr) ν (cm^− 1): 3021, 2401, 1660, 1601, 1415, 1215; ¹H NMR (400 MHz, CDCl₃): δ 8.04 (2H, d, J = 8.48 Hz), 7.86 (2H, s), 7.60 (1H, t, J = 7.4 Hz), 7.51–7.45 (6H, m), 7.0 (4H, d, J = 8.64 Hz), 5.46 (4H, bs), 4.82 (2H, s), 3.88 (6H, s); ¹³C NMR (400 MHz, CDCl₃): δ 195.9, 192.9, 186.1, 160.9, 138.7, 136.1, 133.5, 132.5, 128.6, 128.5, 128.0, 127.0, 114.4, 55.4, 52.1, 45.0; ESI-MS: m/z530 (M + H⁺); HRMS (ESI): m/z calculated for C₃₀H₂₇NO₄S₂ + H⁺ (M + H⁺): 530.1460. Found: 530.1438.

(3E,5E)-2-(4-methoxyphenyl)-2-oxoethyl 3,5-bis(4-methoxybenzylidene)-4-oxopiperidine-1-carbodithioate (11):

Yellow solid (70%); mp 185–188 ^oC; IR (KBr) ν (cm^− 1): 3021, 1661, 1601, 1215, 1023; ¹H NMR (400 MHz, CDCl₃): δ8.02 (2H, d, J = 8.96 Hz), 7.85 (2H, s), 7.46–7.44 (4H, m), 7.01 (4H, d, J = 8.76 Hz), 6.95 (2H, d, J = 8.96 Hz), 5.45 (4H, bs), 4.78 (2H, s), 3.89–3.88 (9H, m); ¹³C NMR (400 MHz, CDCl₃): δ196.1, 191.4, 186.1, 163.8, 160.9, 138.7, 132.5, 130.9, 129.0, 128.0, 127.0, 114.4, 113.8, 55.5, 55.4, 50.6, 44.9; ESI-MS: m/z559 (M + H⁺); HRMS (ESI): m/z calculated for C₃₁H₂₉NO₅S₂ + H⁺ (M + H⁺): 560.1565. Found: 560.1552.

(3E,5E)-benzyl 3,5-bis(3,4-dimethoxybenzylidene)-4-oxopiperidine-1-carbodithioate (12):

Yellow solid (87%); mp 185-187^oC; IR (KBr) ν (cm^− 1): 3021, 1618, 1216, 1146, 670; ¹H NMR (400 MHz, CDCl₃): δ7.83 (2H, s), 7.34–7.26 (6H, m), 7.08 (3H, bs), 6.95 (2H, d, J = 8.28 Hz), 5.44 (4H, bs), 4.53 (2H, s), 3.94–3.91 (12H, m); ¹³C NMR (100 MHz, CDCl₃): δ197.1, 186.1, 150.6, 149.0, 138.8, 135.6, 129.3, 128.5, 127.6, 113.4, 11.2, 56.0, 55.9, 51.4, 42.6; ESI-MS: m/z562 (M + H⁺); HRMS (ESI): m/z calculated for C₃₁H₃₁NO₅S₂ + H⁺ (M + H⁺): 562.1722. Found: 562.1698.

(3E,5E)-methyl 3,5-bis(3,4-dimethoxybenzylidene)-4-oxopiperidine-1-carbodithioate (13):

¹H NMR (400 MHz, CDCl₃): δ7.84 (2H, s), 7.12–7.06 (4H, m), 6.98 (2H, d, J = 8.36Hz), 5.45 (4H, bs), 3.96 (12H, s), 2.61 (3H, s), ¹³C NMR (400 MHz, CDCl₃): δ198.7, 186.1, 150.6, 149.0, 138.7, 128.4, 127.3, 124.4, 113.5, 111.2, 55.9, 51.0, 20.4; ESI-MS: m/z486 (M + H⁺);

(3E,5E)-2-oxo-2-phenylethyl 3,5-bis(3,4-dimethoxybenzylidene)-4-oxopiperidine-1-carbodithioate (14):

Yellow solid (75%); mp 182–185 ^oC; IR (KBr) ν (cm^− 1): 3021, 2401, 1599, 1418, 1216, 1025; ¹H NMR (400 MHz, CDCl₃): δ8.04 (2H, d, J = 8.04 Hz), 7.85 (2H, s), 7.60 (1H, t, J₁ = 7.4 Hz), 7.51–7.47 (2H, m), 7.11–7.05 (4H, m), 6.96 (2H, d, J = 8.28 Hz), 5.45 (4H, bs), 4.83 (2H, s), 3.94 (12H, s), ¹³C NMR (100 MHz, CDCl₃): δ195.9, 192.8, 185.9, 150.6, 149.0, 139.0, 136.1, 133.5, 128.7, 128.4, 127.2, 113.5, 111.2, 56.0, 55.9, 45.0; ESI-MS: m/z590 (M + H⁺); HRMS (ESI): m/z calculated for C₃₂H₃₁NO₆S₂ + H⁺ (M + H⁺): 590.1671. Found: 590.1654.

(3E,5E)-2-(4-methoxyphenyl)-2-oxoethyl 3,5-bis(3,4-dimethoxybenzylidene)-4-oxopiperidine-1-carbodithioate(15):

Yellow solid (78%); mp 171-174^oC; IR (KBr) ν (cm^− 1): 3021, 2401, 1610, 1514, 1217, 1037, 670; ¹H NMR (400 MHz, CDCl₃): δ8.02 (2H, d, J = 8.92 Hz), 7.84 (2H, s), 7.11–7.05 (4H, m), 6.97–6.94 (4H, m), 5.46 (4H, bs), 4.79 (2H, s), 3.95–3.94 (12H, m), 3.88 (3H, s), ¹³C NMR (400 MHz, CDCl₃): δ196.1, 191.2, 185.9, 163.9, 150.6, 149.0, 138.9, 130.8, 129.0, 128.1, 127.2, 124.4, 113.9, 111.2, 56.0, 55.9, 55.5, 50.3, 44.9; ESI-MS: m/z620 (M + H⁺).

(3E,5E)-allyl 3,5-bis(2-methoxybenzylidene)-4-oxopiperidine-1-carbodithioate (16):

IR (KBr) ν (cm^− 1): 3019, 1614, 1487, 1215, 1027; ¹H NMR (500 MHz, CDCl₃): δ 8.06 (2H, s), 7.43–7.39 (2H, m), 7.31 (2H, bs), 7.06–7.03 (2H, m), 6.97 (2H, d, J = 8.32 Hz), 5.88–5.79 (1H, m), 5.26–5.10 (6H, m ), 3.93–3.90 (2H, m), 3.89 (6H, s); ¹³C NMR (100 MHz, CDCl₃):δ196.6, 187.0, 158.3, 134.9, 132.4, 131.2, 130.5, 130.3, 123.5, 120.4, 118.5, 110.9, 55.3, 51.0, 40.5; ESI-MS: m/z452 (M + H⁺); HRMS (ESI): m/z calculated for C₂₅H₂₅NO₃S₂ + H⁺ (M + H⁺): 452.1352. Found:452.1336.

(3E,5E)-benzyl 3,5-bis(2-methoxybenzylidene)-4-oxopiperidine-1-carbodithioate (17)

IR (KBr) ν (cm^− 1): 3019, 2400, 1618, 1215, 1112; ¹H NMR (400 MHz, CDCl₃): δ8.06 (2H, s), 7.40 (2H, t, J = 7.32 Hz),7.37–7.24 (7H, m), 7.04-7.00 (2H, m), 6.95 (2H, d, J = 8.32 Hz), 5.32 (4H, bs), 4.50 (2H, s), 3.87 (6H, s); ¹³C NMR (100 MHz, CDCl₃):δ196.8, 187.1, 158.3, 135.9, 135.0, 131.2, 130.3, 129.2, 128.4, 127.4, 123.5, 120.4, 110.9, 55.5, 50.4, 42.3; ESI-MS: m/z502 (M + H⁺); HRMS (ESI): m/z calculated for C₂₉H₂₇NO₃S₂ + H⁺ (M + H⁺): 502.1511. Found: 502.1479.

(3E,5E)-methyl 3,5-bis(2-methoxybenzylidene)-4-oxopiperidine-1-carbodithioate (18)

IR (KBr) ν (cm^− 1): 3019, 1614, 1462, 1215, 1070; ¹H NMR (400 MHz, CDCl₃): δ8.06 (2H, s), 7.44–7.39 (2H, m), 7.33–7.31 (2H, m), 7.07–7.03 (2H, m), 6.97 (2H, d, J = 8.28 Hz), 5.25 (4H, bs), 3.90 (6H, s), 2.57 (3H, s); ¹³C NMR (100 MHz, CDCl₃):δ198.3, 187.0, 158.3, 134.9, 131.2, 130.6, 130.3, 123.5, 120.4, 110.9, 55.5, 50.6, 20.3; ESI-MS: m/z426 (M + H⁺); HRMS (ESI): m/z calculated for C₂₃H₂₃NO₃S₂ + H⁺ (M + H⁺): 426.1198. Found: 426.1173.

(3E,5E)-2-oxo-2-phenylethyl-3,5-bis(2-methoxybenzylidene)-4-oxopiperidine-1-carbodithioate (19):

IR (KBr) ν (cm^− 1): 3021, 2401, 1612, 1404, 1215, 1075; ¹H NMR (400 MHz, CDCl₃): δ 8.07 (2H, s), 8.03 (2H, d, J = 8.52 Hz), 7.61–7.57 (1H, m), 7.50–7.46 (2H, m), 7.42–7.38 (2H, m), 7.29–7.28 (2H, m), 7.03 (2H, t, J = 7.48 Hz), 6.96 (2H, d, J = 8.28 Hz), 5.32 (4H, bs), 4.79 (2H, s), 3.89 (6H, s); ¹³C NMR (100 MHz, CDCl₃):δ195.4, 193.0, 186.9, 158.3, 136.1, 135.2, 133.4, 131.3, 128.8, 128.6, 128.5, 123.4, 120.5, 111.0, 55.5, 52.3, 44.9; ESI-MS: m/z530 (M + H⁺); HRMS (ESI): m/z calculated for C₃₀H₂₇NO₄S₂ + H⁺ (M + H⁺): 560.1460. Found: 498.1407.

(3E,5E)-2-(4-methoxyphenyl)-2-oxoethyl 3,5-bis(2-methoxybenzylidene)-4-oxopiperidine-1-carbodithioate (20):

IR (KBr) ν (cm^− 1): 3020, 1671, 1603, 1405, 1216, 1028; ¹H NMR (400 MHz, CDCl₃): δ8.06 (2H, s), 8.01 (2H, d, J = 8.92 Hz), 7.42–7.38 (2H, m), 7.31 (2H, bs), 7.05–7.01 (2H, m), 6.97–6.93 (4H, m), 5.37 (4H, bs), 5.32 (2H, s), 3.89–3.88 (9H, m); ¹³C NMR (100 MHz, CDCl₃):δ195.6, 191.5, 186.9, 163.8, 158.3, 135.1, 131.3, 130.8, 130.3, 129.1, 123.4, 120.5, 113.8, 110.9, 55.6, 55.5, 50.1, 44.8; ESI-MS: m/z560 (M + H⁺); HRMS (ESI): m/z calculated for C₃₁H₂₉NO₃S₂ + H⁺ (M + H⁺): 560.1565. Found: 560.1543.

(3E,5E)-allyl 3,5-bis(2-fluorobenzylidene)-4-oxopiperidine-1-carbodithioate (21):

IR (KBr) ν (cm^− 1): 3019, 1622, 1485, 1404, 1215; ¹H NMR (500 MHz, CDCl₃): δ7.92 (2H, s), 7.42–7.40 (4H, m), 7.25–7.22 (2H, m), 7.18–7.14 (2H, m), 5.84–5.76 (1H, m), 5.23–5.08 (6H, m), 3.87 (2H, d, J = 5.36 Hz); ¹³C NMR (100 MHz, CDCl₃):δ197.3, 185.8, 162.0, 159.5, 132.1, 132.1 132.0, 131.7, 131.6, 130.7, 124.3, 124.3, 122.4, 122.2, 118.7, 116.2, 116.0, 50.9, 40.6; ESI-MS: m/z428 (M + H⁺); HRMS (ESI): m/z calculated for C₂₃H₁₉F₂NOS₂ + H⁺ (M + H⁺): 428.0954. Found:428.0944.

(3E,5E)-benzyl 3,5-bis(2-fluorobenzylidene)-4-oxopiperidine-1-carbodithioate (22):

IR (KBr) ν (cm^− 1): 3019, 1621, 1404, 1215; ¹H NMR (500 MHz, CDCl₃): δ7.91 (2H, s), 7.38 (4H, bs), 7.25–7.21 (7H, m), 7.15–7.12 (2H, m), 5.23 (4H, bs), 4.44 (2H, s); ¹³C NMR (100 MHz, CDCl₃):δ197.5, 185.9, 162.0, 159.5, 135.6, 132.1, 132.1, 131.7, 131.6, 130.7, 129.2, 128.5, 127.5, 124.3, 124.3, 122.3, 122.2, 116.2, 116.0, 50.8, 42.5; ESI-MS: m/z478 (M + H⁺); HRMS (ESI): m/z calculated for C₂₇H₂₁F₂NOS₂ + H⁺ (M + H⁺): 478.1111. Found:478.1085.

(3E,5E)-methyl 3,5-bis(2-fluorobenzylidene)-4-oxopiperidine-1-carbodithioate (23):

IR (KBr) ν (cm^− 1): 3019, 2400, 16212, 1405, 1215; ¹H NMR (500 MHz, CDCl₃): δ7.92 (2H, s), 7.41–7.40 (4H, m), 7.25–7.23 (2H, m), 7.18–7.15 (2H, m), 5.25 (4H, bs), 2.53 (3H, s); ¹³C NMR (100 MHz, CDCl₃):δ199.0, 185.8, 162.0, 159.5, 132.2, 132.0, 131.7, 131.6, 130.7, 124.3, 122.4, 122.2, 116.2, 116.0, 50.9, 20.3; ESI-MS: m/z401 (M + H⁺); HRMS (ESI): m/z calculated for C₂₁H₁₇F₂NOS₂ + H⁺ (M + H⁺): 402.0798. Found:402.0786.

(3E,5E)-2-oxo-2-phenylethyl 3,5-bis(2-fluorobenzylidene)-4-oxopiperidine-1-carbodithioate (24):

IR (KBr) ν (cm^− 1): 3019, 2400, 1622, 1404, 1215; ¹H NMR (500 MHz, CDCl₃): δ7.99 (2H, d, J = 5.64 Hz), 7.93 (2H, s), 7.57–7.56 (1H, m), 7.47–7.44 (2H, m), 7.40 (4H, bs), 7.26–7.22 (2H, m), 7.17–7.14 (2H, m), 5.29 (4H, bs), 4.75 (2H, s); ¹³C NMR (100 MHz, CDCl₃):δ196.2, 192.8, 185.6, 162.0, 159.5, 136.0, 133.5, 132.2, 132.2, 131.9,131.8, 131.7, 130.7, 128.7, 128.4, 124.4, 122.3, 122.1, 116.2, 116.0, 51.0, 45.0; ESI-MS: m/z505 (M + H⁺); HRMS (ESI): m/z calculated for C₂₈H₂₁F₂NO₂S₂ + H⁺ (M + H⁺): 506.1060. Found: 506.1043.

(3E,5E)-2-(4-methoxyphenyl)-2-oxoethyl 3,5-bis(2-fluorobenzylidene)-4-oxopiperidine-1-carbodithioate (25):

IR (KBr) ν (cm^− 1): 3019, 2400, 1622, 1404, 1215; ¹H NMR (500 MHz, CDCl₃): δ7.97 (2H, d, J = 6.80 Hz), 7.93 (2H, s), 7.40–7.38 (4H, m), 7.25–7.22 (2H, m), 7.17–7.13 (2H, m), 6.92 (2H, d, J = 6.80 Hz), 5.27 (4H, bs), 4.72 (2H, s), 3.86 (3H, s); ¹³C NMR (100 MHz, CDCl₃):δ196.4, 191.2, 185.6, 163.9, 162.0, 159.5, 132.2, 132.1, 131.9, 131.7, 131.7, 130.8, 130.7, 129.0, 124.4, 122.3, 122.1, 116.2, 116.0, 113.8, 55.5, 51.2, 44.9; ESI-MS: m/z536 (M + H⁺); HRMS (ESI): m/z calculated for C₂₉H₂₃F₂NO₃S₂ + H⁺ (M + H⁺): 536.1166. Found:536.1152.

(3E,5E)-allyl 3,5-bis(4-fluorobenzylidene)-4-oxopiperidine-1-carbodithioate (26):

IR (KBr) ν (cm^− 1): 3021, 1610, 1216, 1157, 670; ¹H NMR (400 MHz, CDCl₃): δ 7.85 (2H, s), 7.49–7.46 (4H, m), 7.18 (4H, t, J = 8.56 Hz), 5.89–5.79 (1H, m), 5.37 (4H, bs), 5.26 (1H, d/d, J₁ = 0.88Hz, J ₂= 16.96 Hz), 5.14 (1H, d, J = 10.12Hz), 3.93 (2H, d, J = 7.00 Hz); ¹³C NMR (100 MHz, CDCl₃): δ197.3, 186.1, 164.6, 162.1, 137.9, 132.4, 132.3, 132.0, 130.5, 129.9, 118.9, 116.2, 116.0, 50.6, 40.7; ESI-MS: m/z428 (M + H⁺); HRMS (ESI): m/z calculated for C₂₃H₁₉F₂NOS₂ + H⁺ (M + H⁺): 428.0954. Found: 428.0950.

(3E,5E)-benzyl 3,5-bis(4-fluorobenzylidene)-4-oxopiperidine-1-carbodithioate (27):

Yellow solid (72%); mp 165–167 ^oC; IR (KBr) ν (cm^− 1): 3021, 2401, 1607, 1509, 1216, 1158, 1023, 670; ¹H NMR (400 MHz, CDCl₃): δ7.84 (2H, s), 7.54–7.46 (4H, m), 7.39–7.35 (1H, m), 7.30–7.28 (4H, m), 7.16 (4H, t, J = 8.4Hz), 5.38 (4H, bs), 4.50 (2H, s); ¹³C NMR (100 MHz, CDCl₃): δ186.2, 164.6, 162.1, 144.0, 137.9, 135.5, 133.2, 133.1, 133.0, 132.4, 132.3, 131.2, 130.4, 130.4, 129.9, 129.4, 129.2, 128.5, 127.6, 126.4, 116.9, 116.7, 116.2, 116.0, 49.9, 42.6; ESI-MS: m/z478 (M + H⁺);

(3E,5E)-2-oxo-2-phenylethyl 3,5-bis(4-fluorobenzylidene)-4-oxopiperidine-1-carbodithioate (28):

Yellow solid (78%); mp 186-188^oC; IR (KBr) ν (cm^− 1): 3021, 2402, 1616, 1509, 1216, 1157, 670; ¹H NMR (400 MHz, CDCl₃): δ 7.94 (2H, d, J = 7.44 Hz), 7.78 (2H, d), 7.51 (1H, t, J = 7.4 Hz), 7.42–7.36 (6H, m), 7.08 (4H, t, J = 8.45 Hz), 5.32 (4H, bs), 4.71 (2H, s); ¹³C NMR (100 MHz, CDCl₃): δ196.2, 192.7, 186.0, 164.6, 162.1, 138.1, 136.0, 133.6, 1332.5, 132.4, 130.4, 130.3, 129.7, 128.7, 128.4, 116.3, 116.1, 50.7, 45.1; ESI-MS: m/z506 (M + H⁺); HRMS (ESI): m/z calculated for C₂₈H₂₁F₂NO₂S₂ + H⁺ (M + H⁺): 506.1060. Found: 506.1058.

(3E,5E)-2-(4-methoxyphenyl)-2-oxoethyl 3,5-bis(4-fluorobenzylidene)-4-oxopiperidine-1-carbodithioate (29):

Yellow solid (74%); mp 177–179 ^oC; IR (KBr) ν (cm^− 1): 3021, 2401, 1660, 1605, 1491, 1216, 1096, 1025; ¹H NMR (400 MHz, CDCl₃): δ 8.01 (2H, d, J = 8.96 Hz), 7.86 (2H, s), 7.49–7.45 (4H, m), 7.17 (4H, t, J = 8.58 Hz), 6.95 (2H, d, J = 8.96 Hz), 5.41 (4H, bs), 4.77 (2H, s), 3.89 (3H, s); ¹³C NMR (100 MHz, CDCl₃): δ196.4, 191.1, 186.0, 164.6, 163.9, 162.1, 138.1, 132.4, 132.4, 130.8, 130.4, 129.7, 129.0, 116.3, 116.0, 113.9, 55.5, 51.0, 44.9; ESI-MS: m/z536 (M + H⁺); HRMS (ESI): m/z calculated for C₂₉H₂₃F₂NO₃S₂ + H⁺ (M + H⁺): 536.1166. Found: 536.1152.

(3E,5E)-allyl 3,5-bis(4-chlorobenzylidene)-4-oxopiperidine-1-carbodithioate (30):

Yellow solid (70%); mp 173-175^oC; IR (KBr) ν (cm^− 1): 3021, 2401, 1616, 1216, 1096; ¹H NMR (400 MHz, CDCl₃ ): δ 7.84 (2H, s), 7.48–7.40 (8H, m), 5.90–5.79 (1H, m), 5.37 (4H, bs), 5.27–5.24 (1H, m), 5.15 (1H, d, J = 10 Hz), 3.93 (2H, d, J = 6.96 Hz); ¹³C NMR (100 MHz, CDCl₃): δ197.4, 185.9, 137.8, 136.0, 132.7, 131.9, 131.5, 130.5, 129.2, 118.9, 50.6, 40.7; ESI-MS: m/z459 (M + H⁺); HRMS (ESI): m/z calculated for C₂₃H₁₉Cl₂NOS₂ + H⁺ (M + H⁺): 460.0363. Found: 460.0352.

(3E,5E)-methyl 3,5-bis(4-chlorobenzylidene)-4-oxopiperidine-1-carbodithioate (31):

IR (KBr) ν (cm^− 1): 3021, 2401, 1620, 1216, 670; ¹H NMR (400 MHz, CDCl₃ ): δ 7.84 (2H, s), 7.47 (4H, d, J = 8.56 Hz), 7.41 (4H, d, J = 8.48 Hz), 5.38 (4H, bs), 2.59 (3H, s); ¹³C NMR (100 MHz, CDCl₃): δ 199.2, 185.9, 137.8, 135.9 132.7, 131.5, 130.6, 129.2, 50.6, 20.4; ESI-MS: m/z433 (M + H⁺); HRMS (ESI): m/z calculated for C₂₁H₁₇Cl₂NOS₂ + H⁺ (M + H⁺): 434.0207. Found: 434.0189.

(3E,5E)-2-oxo-2-phenylethyl 3,5-bis(4-chlorobenzylidene)-4-oxopiperidine-1-carbodithioate (32):

Yellow solid (72%); mp 162–165 ^oC; IR (KBr) ν (cm^− 1): 3021, 2401, 1680, 1614, 1215, 1095; ¹H NMR (400 MHz, CDCl₃ ): δ8.03 (2H, d, J = 8.16 Hz), 7.61 (1H, t, J = 7.40 Hz), 7.51–7.40 (10H, m), 5.40 (4H, bs), 4.81 (2H, s); ¹³C NMR (100 MHz, CDCl₃): δ196.3, 192.6, 185.8, 138.0, 136.0, 133.6, 132.6, 131.5, 129.2, 128.7, 128.4, 51.4, 45.1; ESI-MS: m/z538 (M + H⁺); HRMS (ESI): m/z calculated for C₂₁H₁₇Cl₂NOS₂ + H⁺ (M + H⁺): 538.0469. Found: 538.0458.

(3E,5E)-2-(4-methoxyphenyl)-2-oxoethyl 3,5-bis(4-chlorobenzylidene)-4-oxopiperidine-1-carbodithioate (33):

Yellow solid (76%); mp 178-180^oC; IR (KBr) ν (cm^− 1): 3021, 2401, 1672, 1610, 1491, 1216, 1096, 1025; ¹H NMR (400 MHz, CDCl₃ ): δ8.00 (2H, d, J = 8.92 Hz), 7.83 (2H, s), 7.44 (4H, d, J = 8.56 Hz), 7.39 (4H, d, J = 8.56 Hz), 6.95 (2H, d, J = 8.92 Hz), 5.38 (4H, bs), 4.76 (2H, s), 3.88 (3H, s); ¹³C NMR (100 MHz, CDCl₃): δ 196.5, 191.1, 185.8, 163.9, 138.0, 136.0, 132.6, 131.5, 130.8, 129.2, 113.9, 55.5, 51.6, 44.9; ESI-MS: m/z568 (M + H⁺); HRMS (ESI): m/z calculated for C₂₉H₂₃Cl₂NO₃S₂ + H⁺ (M + H⁺): 568.0575. Found: 568.0567.

(3E,5E)-benzyl 3,5-bis(4-methylbenzylidene)-4-oxopiperidine-1-carbodithioate (34):

Yellow solid (80%); mp 146-150^oC; IR (KBr) ν (cm^− 1): 3020, 2402, 1609, 1512, 1216, 1024, 670; ¹H NMR (400 MHz, CDCl₃): δ 7.86 (2H, s), 7.38–7.36 (4H, m), 7.31–7.25 (9H, m), 5.41 (4H, bs), 4.50 (2H, s), 2.41 (6H, s); ¹³C NMR (400 MHz, CDCl₃): δ 197.3, 186.6, 140.2, 139.0, 135.7, 131.5, 130.5, 129.6, 129.4, 129.3, 128.5, 127.4, 58.4, 51.05, 42.5; ESI-MS: m/z469 (M + H⁺);

(3E,5E)-methyl 3,5-bis(4-methylbenzylidene)-4-oxopiperidine-1-carbodithioate (35):

¹H NMR (400 MHz, CDCl₃): δ7.88 (2H, s), 7.39 (4H, d, J = 7.88 Hz), 7.30 (4H, d, J = 8.00 Hz), 5.42 (4H, bs), 2.58 (3H, s), 2.43 (6H, s); ¹³C NMR (400 MHz, CDCl₃): δ 198.9, 186.4, 140.2, 138.9, 131.6, 130.5, 129.6, 129.5, 50.9, 21.5, 20.4; ESI-MS: m/z394 (M + H⁺); HRMS (ESI): m/z calculated for C₂₃H₂₃NOS₂ + H⁺ (M + H⁺): 394.1299. Found: 394.1302.

(3E,5E)-2-oxo-2-phenylethyl 3,5-bis(4-methylbenzylidene)-4-oxopiperidine-1-carbodithioate (36):

Yellow solid (78%); mp 162–164 ^oC; IR (KBr) ν (cm^− 1): 3021, 2401, 1612, 1513, 1413, 1215, 670; ¹H NMR (400 MHz, CDCl₃): δ8.03 (2H, d, J = 8.36 Hz), 7.88 (2H, s), 7.62–7.57 (1H, m), 7.50–7.46 (2H, m), 7.38 (4H, d, J = 7.96Hz), 7.28 (4H, d, J = 7.96 Hz), 5.44 (4H, bs), 4.80 (2H, s), 2.42 (6H, s); ¹³C NMR (400 MHz, CDCl₃): δ 196.0, 192.9, 186.3, 140.3, 139.2, 136.1, 133.5, 131.5, 130.5, 129.6, 129.2, 128.6, 128.5, 51.8, 45.0, 21.5; ESI-MS: m/z498 (M + H⁺); HRMS (ESI): m/z calculated for C₃₀H₂₇NO₂S₂ + H⁺ (M + H⁺): 498.1517. Found: 498.1545.

(3E,5E)-2-(4-methoxyphenyl)-2-oxoethyl 3,5-bis(4-methylbenzylidene)-4-oxopiperidine-1-carbodithioate (37):

Yellow solid (77%); mp 170-172^oC; IR (KBr) ν (cm^− 1): 3020, 2401, 1605, 1216, 670; ¹H NMR (400 MHz, CDCl₃): δ 8.01 (2H, d, J 8.80 Hz), 7.86 (2H, s),7.36 (4H,d, J = 7.72 Hz), 7.26 (4H, d, J = 7.96 Hz), 6.93 (2H, d, J = 8.83 Hz), 5.45 (4H, bs), 4.75 (2H, s), 3.87 (3H, s), 2.40 (6H, s); ¹³C NMR (400 MHz, CDCl₃): δ196.2, 191.5, 186.3, 163.8, 140.3, 139.2, 131.5, 130.9, 130.5, 129.6, 129.2, 129.0, 113.8, 55.5, 51.4, 44.8, 21.5; ESI-MS: m/z528 (M + H⁺); HRMS (ESI): m/z calculated for C₃₁H₂₉NO₃S₂ + H⁺ (M + H⁺): 528.1667. Found: 528.1660.

(3E,5E)-allyl 3,5-bis(4-isopropylbenzylidene)-4-oxopiperidine-1-carbodithioate (38):

IR (KBr) ν (cm^− 1): 3019, 2964, 1611, 1509, 1215; ¹H NMR (400 MHz, CDCl₃): δ7.87 (2H, s), 7.44 (4H, d, J = 8.00 Hz), 7.35 (4H, d, J = 8.24 Hz), 5.91–5.81 (1H, m), 5.43 (4H, s), 5.28 (1H, d/d, J₁ = 1.32 Hz, J ₂= 2.72 Hz), 5.24–5.12 (1H, m), 3.95–3.93 (2H, m), 3.02–2.95 (2H, m), 1.30 (12H, d, J = 6.92 Hz); ¹³C NMR (100 MHz, CDCl₃): δ197.0, 186.4, 151.0, 138.9, 132.2, 131.9, 130.7, 129.4, 127.0, 118.7, 50.9, 40.7, 34.0, 23.7; ESI-MS: m/z476 (M + H⁺); HRMS (ESI): m/z calculated for C₂₉H₃₃NOS₂ + H⁺ (M + H⁺): 476.2082. Found: 476.2058.

(3E,5E)-benzyl 3,5-bis(4-isopropylbenzylidene)-4-oxopiperidine-1-carbodithioate (39):

IR (KBr) ν (cm^− 1): 3019, 1612, 1413, 1215; NMR (400 MHz, CDCl₃): δ7.87 (2H, s), 7.43–7.42 (4H, m), 7.34–7.27 (9H, m), 5.42 (4H, s), 4.52 (2H, s), 3.02–2.91 (2H, m), 1.29 (12H, d, J = 6.92 Hz); ¹³C NMR (100 MHz, CDCl₃): δ197.2, 186.5, 151.0, 138.9, 135.7, 131.9, 130.7, 129.5, 129.3, 128.5, 127.5, 127.0, 50.7, 42.6, 34.0, 23.7; ESI-MS: m/z526 (M + H⁺); HRMS (ESI): m/z calculated for C₃₃H₃₅NOS₂ + H⁺ (M + H⁺): 526.2238. Found: 526.2203.

(3E(3E,5E)-methyl 3,5-bis(4-isopropylbenzylidene)-4-oxopiperidine-1-carbodithioate (40):

IR (KBr) ν (cm^− 1): 3021, 2401, 1612, 1403, 1215; NMR (400 MHz, CDCl₃): δ7.88 (2H, s), 7.44 (4H, d, J = 8.08 Hz), 7.35 (4H, d, J = 8.20 Hz), 5.43 (4H, bs), 3.04–2.93 (2H, m), 2.60 (3H, s), 1.31 (12H, d, J = 6.92 Hz); ¹³C NMR (100 MHz, CDCl₃): δ198.9, 186.5, 151.0, 138.9, 131.9, 130.7, 129.5, 127.0, 50.9, 34.0, 23.7, 20.4; ESI-MS: m/z450 (M + H⁺); HRMS (ESI): m/z calculated for C₂₇H₃₁NOS₂ + H⁺ (M + H⁺): 450.1925. Found: 450.1914.

(3E,5E)-2-oxo-2-phenylethyl 3,5-bis(4-isopropylbenzylidene)-4-oxopiperidine-1-carbodithioate (41):

IR (KBr) ν (cm^− 1): 3019, 1660, 1612, 1215; ¹H NMR (400 MHz, CDCl₃): δ8.04 (2H, d, J = 8.52 Hz), 7.89 (2H, s), 7.62–7.58 (1H, m), 7.51–7.47 (2H, m), 7.43 (4H, d, J = 8.04 Hz), 7.33 (4H, d, J = 8.20 Hz), 5.46 (4H, s), 4.81 (2H, s), 3.02–2.92 (2H, m), 1.30 (12H, d, J = 6.92 Hz); ¹³C NMR (100 MHz, CDCl₃): δ196.0, 192.9, 186.2, 151.1, 139.1, 136.1, 133.5, 131.8, 130.7, 129.2, 128.7, 128.5, 127.0, 51.4, 45.0, 34.0, 23.7; ESI-MS: m/z554 (M + H⁺); HRMS (ESI): m/z calculated for C₃₄H₃₅NO₂S₂ + H⁺ (M + H⁺): 554.2187. Found: 554.2167.

(3E,5E)-2-(4-methoxyphenyl)-2-oxoethyl 3,5-bis(4-isopropylbenzylidene)-4-oxopiperidine-1-carbodithioate (42):

IR (KBr) ν (cm^− 1): 3021, 1660, 1612, 1215; ¹H NMR (400 MHz, CDCl₃): δ8.02 (2H, d, J = 8.92 Hz), 7.88 (2H, s), 7.43 (4H, d, J = 8.00 Hz), 7.33 (4H, d, J = 8.20 Hz), 6.95 (2H, d, J = 8.96 Hz), 5.46 (4H, s), 4.78 (2H, s), 3.88 (3H, s), 3.02–2.92 (2H, m), 1.29 (12H, d, J = 6.92 Hz); ¹³C NMR (100 MHz, CDCl₃): δ196.2, 191.4, 186.3, 163.8, 151.1, 139.1, 131.8, 130.9, 130.7, 129.2, 129.0, 127.0, 113.8, 55.5, 51.2, 44.9, 34.0, 23.7; ESI-MS: m/z584 (M + H⁺); HRMS (ESI): m/z calculated for C₃₅H₃₇NO₃S₂ + H⁺ (M + H⁺): 584.2293. Found: 584.2250.

(3E,5E)-allyl 3,5-bis(2-methylbenzylidene)-4-oxopiperidine-1-carbodithioate (43):

IR (KBr) ν (cm^− 1): 3019, 1618, 1413, 1215; ¹H NMR (500 MHz, CDCl₃): δ8.01 (2H, s), 7.33–7.21 (8H, m), 5.82–5.75 (1H, m), 5.22–5.07 (6H, m), 3.87–3.85 (2H, m), 2.36 (6H, s); ¹³C NMR (100 MHz, CDCl₃):δ196.9, 186.8, 138.6, 138.2, 133.4, 132.2 130.9, 130.5, 129.6, 128.8, 125.9, 118.6, 50.8, 40.5, 20.1; ESI-MS: m/z420 (M + H⁺); HRMS (ESI): m/z calculated for C₂₅H₂₅NOS₂ + H⁺ (M + H⁺): 420.1456. Found:420.1453.

((3E,5E)-benzyl 3,5-bis(2-methylbenzylidene)-4-oxopiperidine-1-carbodithioate (44):

IR (KBr) ν (cm^− 1): 3019, 2400, 1617, 1385, 1215; ¹H NMR (500 MHz, CDCl₃): δ8.00 (2H, s), 7.30–7.22 (13H, m), 5.17 (4H, bs), 4.44 (2H, s), 2.33 (6H, s); ¹³C NMR (100 MHz, CDCl₃):δ197.1, 186.9, 138.6, 138.2, 135.8, 133.4, 130.9, 130.5, 129.5, 129.2, 128.8, 128.5, 127.5, 125.9, 50.6, 42.3, 20.1; ESI-MS: m/z470 (M + H⁺); HRMS (ESI): m/z calculated for C₂₉H₂₇NOS₂ + H⁺ (M + H⁺): 470.1612. Found:470.1605.

(3E,5E)-methyl 3,5-bis(2-methylbenzylidene)-4-oxopiperidine-1-carbodithioate (45):

IR (KBr) ν (cm^− 1): 3019, 1615, 1215; ¹H NMR (500 MHz, CDCl₃): δ8.01 (2H, s), 7.31–7.21 (8H, m), 5.18 (4H, bs), 2.52 (3H, s), 2.36 (6H, s); ¹³C NMR (100 MHz, CDCl₃):δ198.7, 186.7, 138.5, 138.2, 133.4, 130.9, 130.5, 129.6, 128.8, 125.9, 50.8, 20.3, 20.1; ESI-MS: m/z394 (M + H⁺); HRMS (ESI): m/z calculated for C₂₃H₂₃NOS₂ + H⁺ (M + H⁺): 394.1299. Found: 394.1289.

(3E,5E)-2-oxo-2-phenylethyl 3,5-bis(2-methylbenzylidene)-4-oxopiperidine-1-carbodithioate (46):

IR (KBr) ν (cm^− 1): 3020, 1680, 1614, 1407, 1216; ¹H NMR (400 MHz, CDCl₃): δ8.06 (2H, s), 8.01 (2H, d, J = 8.04 Hz), 7.59 (1H, t, J = 7.40 Hz), 7.47 (2H, t, J = 7.88 Hz), 7.34–7.22 (8H, m), 5.24 (4H, bs), 4.76 (2H, s), 2.38 (6H, s); ¹³C NMR (100 MHz, CDCl₃):δ195.7, 192.8, 186.6, 138.8, 138.2, 136.1, 133.5, 133.3, 130.5, 129.6, 128.8, 128.6, 128.4, 125.9, 52.1, 50.2, 44.9, 20.1; ESI-MS: m/z498 (M + H⁺); HRMS (ESI): m/z calculated for C₃₀H₂₇NO₂S₂ + H⁺ (M + H⁺): 498.1561. Found: 498.1535.

(3E,5E)-2-(4-methoxyphenyl)-2-oxoethyl 3,5-bis(2-methylbenzylidene)-4-oxopiperidine-1-carbodithioate (47)

IR (KBr) ν (cm^− 1): 3018, 1601, 1215; ¹H NMR (400 MHz, CDCl₃): δ8.05 (2H, s), 8.00 (2H, d, J = 8.96 Hz), 7.34–7.24 (8H, m), 6.94 (2H, d, J = 8.96 Hz), 5.24 (4H, bs), 4.74 (2H, s), 3.88 (3H, s), 2.838 (6H, s); ¹³C NMR (100 MHz, CDCl₃):δ196.0, 191.3, 186.6, 163.8, 138.7, 138.2, 133.3, 130.8, 130.7, 130.6, 129.6, 128.8, 125.9, 113.8, 55.5, 50.6, 44.8, 20.1; ESI-MS: m/z528 (M + H⁺); HRMS (ESI): m/z calculated for C₃₁H₂₉NO₃S₂ + H⁺ (M + H⁺): 528.1667. Found:528.1621.

(3E,5E)-allyl 3,5-bis(3-methylbenzylidene)-4-oxopiperidine-1-carbodithioate (48):

¹H NMR (500 MHz, CDCl₃): δ7.85 (2H, s), 7.37–7.33 (2H, m), 7.27–7.22 (6H, m), 5.87–5.79 (1H, m), 5.37–5.21 (5H, m), 5.12–5.09 (2H, m), 5.91 (2H, d, J = 5.56 Hz), 2.40 (6H, s); ¹³C NMR (125 MHz, CDCl₃): δ197.0, 186.4, 139.2, 138.5, 134.3, 132.2, 131.2, 130.6, 130.1, 128.7, 127.3, 118.7, 50.9, 40.7, 21.4; ESI-MS: m/z420 (M + H⁺); HRMS (ESI): m/z calculated for C₂₅H₂₅NOS₂ + H⁺ (M + H⁺): 420.1456. Found: 420.1438.

(3E,5E)-benzyl 3,5-bis(3-methylbenzylidene)-4-oxopiperidine-1-carbodithioate (49):

IR (KBr) ν (cm^− 1): 3019, 2400, 1614, 1215, 1070; ¹H NMR (500 MHz, CDCl₃): δ7.84 (2H, s), 7.34–7.20 (13H, m), 5.37 (4H, bs), 4.49 (2H, s), 2.37 (6H, s); ¹³C NMR (125 MHz, CDCl₃): δ197.3, 186.5, 139.2, 138.5, 135.8, 134.3, 131.2, 130.6, 130.1, 129.3, 128.7, 128.5, 127.5, 127.3, 50.5, 42.5, 21.4; ESI-MS: m/z470 (M + H⁺); HRMS (ESI): m/z calculated for C₂₉H₂₇NOS₂ + H⁺ (M + H⁺): 470.1612. Found: 470.1596.

(3E(3E,5E)-methyl 3,5-bis(3-methylbenzylidene)-4-oxopiperidine-1-carbodithioate (50):

¹H NMR (500 MHz, CDCl₃): δ7.85 (2H, s), 7.35 (2H, t, J = 6.08 Hz), 7.27 (3H, s), 7.25–7.22 (3H, m), 5.38 (4H, bs), 2.56 (3H, s), 2.41 (6H, s); ¹³C NMR (100 MHz, CDCl₃): δ198.8, 186.4, 139.2, 138.5, 134.3, 131.2, 130.6, 130.2, 128.7,127.3, 50.9, 21.4, 20.4; ESI-MS: m/z394 (M + H⁺); HRMS (ESI): m/z calculated for C₂₃H₂₃NOS₂ + H⁺ (M + H⁺): 394.1299. Found: 394.1291.

(3E,5E)-2-oxo-2-phenylethyl 3,5-bis(3-methylbenzylidene)-4-oxopiperidine-1-carbodithioate (51):

IR (KBr) ν (cm^− 1): 3020, 1614, 1404, 1215, 1070; ¹H NMR (500 MHz, CDCl₃): δ 8.01 (2H, d, J = 6.44 Hz), 7.86 (2H, s), 7.58–7.55 (1H, m), 7.47–7.44 (2H, m), 7.34 (2H, t, J = 6Hz), 7.26–7.21 (6H, m), 5.41 (4H, bs), 4.77 (2H, s), 2.39 (6H, s); ¹³C NMR (100 MHz, CDCl₃):δ196.0, 192.8, 186.2, 139.4, 138.5, 136.1, 134.2, 133.5, 131.2, 130.7, 129.9, 128.7, 128.7, 128.5, 127.3, 51.8, 45.0, 21.4; ESI-MS: m/z498 (M + H⁺); HRMS (ESI): m/z calculated for C₃₀H₂₇NO₂S₂ + H⁺ (M + H⁺): 498.1561. Found: 560.1543.

(3E,5E)-2-(4-methoxyphenyl)-2-oxoethyl 3,5-bis(3-methylbenzylidene)-4-oxopiperidine-1-carbodithioate (52):

IR (KBr) ν (cm^− 1): 3018, 1668, 1602, 1408, 1218, 1028; ¹H NMR (500 MHz, CDCl₃): δ8.16 (2H, d, J = 7.16 Hz),, 7.85 (2H, s), 7.34 (2H, t, J = 6.04 Hz), 7.26–7.21 (6H, m), 6.92 (2H, d, J = 7.16 Hz), 5.41 (4H, bs), 4.74 (2H, s), 3.86 (3H, s), 2.39 (6H, s); ¹³C NMR (100 MHz, CDCl₃):δ196.2, 191.3, 186.3, 163.8, 139.4, 138.5, 134.2, 131.2, 130.8, 130.6, 130.0, 129.0, 128.7, 127.3, 113.8, 55.5, 51.4, 44.8, 21.4; ESI-MS: m/z528 (M + H⁺); HRMS (ESI): m/z calculated for C₃₁H₂₉NO₃S₂ + H⁺ (M + H⁺): 528.1667. Found:528.1652.

Biology

In vitro DNA ligation assay

For the study of antiligase activity of compounds the in vitro DNA ligation assay was performed as described previously (Krishna et al., 2014). Briefly, the three different DNA oligos, 52 Mer (5‘-GTACGTCGATCGATTGGTAGATCAGGGTCTATGTATGTCAGTGAGATAGTAC-3‘), 25 Mer (5′-CTGATCTACCAATCGATCGACGTAC-3′) and 27 mer (5′/5Cy3/GTACTATCTC ACTGACATACATAGACA-3′) were annealed for the generation of nicked DNA substrate for ligase I protein. During the ligation assay, different concentration of compounds was incubated with hLig1p and DNA in a ligation buffer at 37°C for 30 minutes. The reaction mixtures were run in denaturing gel containing urea and gel images were captured and the band density were calculated by Image Quant LAS 4010. In the gel the ligated DNA (52 Mer) was observed at the top of the gel whereas unligated DNA (27 Mer) substrate appeared lower on the gel.

Antiproliferative assay

As described previously [29, 25], the MTT assay was performed to measure the cytotoxicity of compounds in DLD-1 versus normal HEK-293 cells. Briefly, the colon cancer cell line DLD-1 and human embryonic kidney cells HEK-293 (non-cancerous cells) were seeded in 96 well plates and incubated overnight. The cells were treated with different concentrations of compounds for 48h and then MTT was added in each well and percentage of dead cells was calculated.

Clonogenic cell survival assay

The clonogenic cell survival assay was performed as described previously [27]. Briefly, DLD-1 cells (100–200 cells/well) were grown in 24 well culture plates and treated with different concentration of hLig1 inhibitors for 24 and 48 h. After the treatment culture medium was replaced with fresh medium and cells were cultured again for another 15 days while changing the media every 3 days. Cells were washed with 1XPBS, and stained with crystal violet. The excess stain was removed by washing with distilled water and the image from each well was captured.

Docking study

To identify the mode of binding of the inhibitors with hLigI, we conducted in silico docking studies for all the active compounds. The compounds were docked to the crystal structure of hLig1p-DNA complex. We have retrieved the crystal structure of hLigI from the PDB database (ID:1X9N) [14]. Further, structure preparation was performed prior to docking. For this step, the nicked DNA with Adenosine Monophosphate (AMP) co-crystalized structure was removed. Subsequently, hydrogen atoms were added to the protein. Finally, energy minimization was performed using the Protein preparation wizard of Schrödinger suite of software (Schrödinger Release 2020-3: Protein Preparation Wizard; Schrödinger, LLC, New York, NY, 2016.). The active site remained same as described in our previous study [28]. The structures of the inhibitors were drawn, and energy minimized using LigPrep module incorporated in Schrödinger (Schrödinger Release 2020-3: LigPrep, Schrödinger, LLC, New York, NY, 2020). All the docking experiments were performed using Glide module of Schrödinger (Schrödinger Release 2020-3: Glide, Schrödinger, LLC, New York, NY, 2020).

Cell cycle analysis

For analysis of cell cycle arrest, cells were synchronized by serum starvation for 36 h and afterwards grown in media containing serum in the presence or absence of compound 49 at 3 µM concentration. After every 8h interval, cells were harvested, washed with cold PBS twice and fixed with chilled 70% ethanol. The fixed cells were further processed by washing with chilled PBS and then staining was done by 50 µg/mL of propidium iodide. Cell cycle distribution was analyzed by FACS-Calibur (BD Biosciences, SanDiago, CA).

Acknowledgement

We acknowledge CSIR-CDRI, Govt. of India, for financial and infrastructural support. Financial support from SERB grant (EMR/2017/002080) is acknowledged. Authors also acknowledge CSIR (D.M., DKS), DBT (PM), UGC(SK), and ICMR (SK) for research fellowships. We acknowledge Mrs. Tara Rawat (Senior Technical Officer) for technical assistance and SAIF Division for acquiring NMR spectral data. This manuscript bears the CDRI Manuscript No.14/2020/DB.

Declaration of interest

Authors declare that there is no conflict of interest.

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Schemes 1 and 2 are available in the Supplementary Files section

Scheme1.jpg
Previously reported hLigI inhibitors (a-g) along with the scheme of newly synthesized molecules (h).
Scheme2.jpg
Reagents and conditions: (a) Glacial acetic acid, conc. HCl, RT, 48 h; (b) K₂CO₃, acetone:water (5:1), 20 ^oC, 2 h; (c) carbon disulfide (1.3 equiv), aqNaOH, DCM, 0 ^oC, 2 h; (d) R-Br (1.2 equiv.), water:acetone (5:1), 10-15^oC, 1–2 h.
SupportingfilechemistryMedicinalChemistryResearch.docx

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Improved hLigI inhibition activity of Curcumin Monocarbonyl Analogue-Dithiocarbamate Hybrids: Design, Synthesis and Biology

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Abstract

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1. Introduction

2. Results And Discussion

2.1 Chemistry

2.2 Biological Evaluation

2.3 In silico Binding Study of Compounds with hLigI Protein

2.4 Cell cycle analysis of DLD-1 after treatment with compound 49

3. Conclusions

Experimental Section

Declarations

References

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Supplementary Files

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